1. Field of the Invention
The invention relates to novel active principles for vaccines derived from the major surface protein in merozoite forms of a Plasmodium which is infectious for mammals, especially humans, more generally termed MSP-1.
2. Discussion of the Background
MSP-1 has already been the subject of a number of studies. It is synthesised in the schizont stage of Plasmodium type parasites, in particular Plasmodium falciparum, and is expressed in the form of one of the major surface constituents of merozoites both in the hepatic stage and in the erythrocytic stage of malaria (1, 2, 3, 4). Because of the protein's predominant character and conservation in all known Plasmodium species, it has been suggested that it could be a candidate for constituting anti-malarial vaccines (5, 6).
The same is true for fragments of that protein, particularly the natural cleavage products which are observed to form, for example during invasion by the parasite into erythrocytes of the infected host. Among such cleavage products are the C-terminal fragment with a molecular weight of 42 kDa (7, 8) which is itself cleaved once more into an N-terminal fragment with a conventional apparent molecular weight of 33 kDa and into a C-terminal fragment with a conventional apparent molecular weight of 19 kDa (9) which remains normally fixed to the parasite membrane after the modifications carried out on it, via glycosylphosphatidylinositol (GPI) groups (10, 11).
It is also found at the early ring stage of the intraerythrocytic development cycle (15, 16), whereby the observation was made that the 19 kDa fragment could play a role which is not yet known, but which is doubtless essential in re-invasive processes. This formed the basis for hypotheses formed in the past that that protein could constitute a particularly effective target for possible vaccines.
It should be understood that the references frequently made below to the p42 and p19 proteins from a certain type of Plasmodium are understood to refer to the corresponding C-terminal cleavage products of the MSP-1 protein of that Plasmodium or, by extension, to products containing substantially the same amino acid sequences, obtained by genetic recombination or by chemical synthesis using conventional techniques, for example using the “Applied System” synthesiser, or by “Merrifield” type solid phase synthesis. For convenience, references to “recombinant p42” and “recombinant p19” refer to “p42” and “p19” obtained by techniques comprising at least one genetic engineering step.
Faced with the difficulty of obtaining large quantities of parasites for P. falciparum and the impossibility of cultivating P. vivax in vitro, it has become clear that the only means of producing an anti-malaria vaccine is to resort to techniques which use recombinant proteins or peptides. However, MSP-1 is very difficult to produce whole because of it large size of about 200 kDa, a fact which has led researchers to study the C-terminal portion, the (still unknown) function of which is probably the more important.
Recombinant proteins concerning the C-terminal portion of the P. falciparum MSP-1 which have been produced and tested in the monkey (12, 40, 41) are:                a p19 fused with a glutathione-S-transferase produced in E. coli (40);        a p42 fused with a glutathione-S-transferase produced in E. coli (12);        a p19 fused with a polypeptide from a tetanic anatoxin carrying auxiliary T cell epitopes produced in S. cerevisiae (12);        a p42 product in a baculovirus system (41).        
A composition containing a p19 protein fused with a glutathione-S-transferase produced in E. coli combined with alum or liposomes did not exhibit a protective effect in any of six vaccinated Aotus nancymai monkeys (40).
A composition containing a p42 protein fused with a glutathione-S-transferase produced in E. coli combined with a Freund complete adjuvant did not exhibit a protective effect in two types of Aotus monkeys (A. nancymai and A. vociferans) when administered to them. The p19 protein produced in S. cerevisiae exhibited a protective effect in two A. nancymai type Aotus monkeys (12). In contrast, there was no protective effect in two A. vociferans type Aotus monkeys.
Some researchers (Chang et al.) have also reported immunisation tests carried out in the rabbit using a recombinant p42 protein produced in a baculovirus system and containing one amino acid sequence in common with P. falciparum (18). Thus these latter authors indicate that in the rabbit that recombinant p42 behaves substantially in the same way as the entire recombinant MSP-1 protein (gp195). This p42 protein in combination with a Freund complete adjuvant has been the subject matter of a vaccination test in a non-human primate susceptible to infection by P. falciparum, Aotus, lemurinus grisemembra (40). The results showed that 2 of 3 animals were completely protected and the third, while exhibiting a parasitemia which resembled that of the controls, had a longer latent period. It is nevertheless risky to conclude to a protective nature in man of the antibodies thus induced against the parasites themselves. It should be remembered that there are currently no very satisfactory experimental models in the primate for P. vivax and P. falciparum. The Saimiri model, developed for P. falciparum and P. vivax, and the Aotus model for P. falciparum, are artificial systems requiring the parasite strains to be adapted and often requiring splenectomy of the animals to obtain significant parasitemia. As a result, the vaccination results from such models can only have a limited predictive value for man.
In any event, what the real vaccination rate would be which could possibly be obtained with such recombinant proteins is also questionable, bearing in mind the discovery—reported below—of the presence in p42s from Plasmodiums of the same species, and more particularly in the corresponding p33s, of hypervariable regions which would in many cases render uncertain the immunoprotective efficacy of antibodies induced in individuals vaccinated with a p42 from a Plasmodium strain against an infection by other strains of the same species (13).
It can even be assumed that the high polymorphism of the N-terminal portion of p42 plays a significant role in immune escape, often observed for that type of parasite.
The aim of the present invention is to produce vaccinating recombinant proteins which can escape these difficulties, the protective effect of which is verifiable in genuinely significant experimental models or even directly in man.
More particularly, the invention provides vaccinating compositions against a Plasmodium type parasite which is infectious for man, containing as an active principle a recombinant protein which may or may not be glycosylated, whose essential constituent polypeptide sequence is:                either that of a 19 kilodalton (p19) C-terminal fragment of the surface protein 1 of the merozoite form (MSP-1 protein) of a Plasmodium type parasite which is infectious for man, said C-terminal fragment remaining normally anchored to the parasite surface at the end of its penetration phase into human erythrocytes in the event of an infectious cycle;        or that of a portion of that fragment which is also capable of inducing an immune response which can inhibit in vivo parasitemia due to the corresponding parasite;        or that of an immunologically equivalent peptide of said p19 fragment or said portion of that fragment; andsaid recombinant protein further comprises conformational epitopes which are unstable in a reducing medium and which preferably constitute the majority of the epitopes recognised by human antiserums formed against the corresponding Plasmodium.         